Sudden cardiac death due to sustained ventricular arrhythmias continues to be a major health care problem. Abnormal intracellular Ca cycling has been implicated in the pathogenesis of heart diseases including congenital and acquired cardiac arrhythmias. However the specific mechanisms linking abnormal Ca handling and arrhythmogenesis remain to be elucidated. On a beat-to-beat basis, intracellular Ca release through sarcoplasmic reticulum (SR) cardiac ryanodine receptor (RyR2) channels is elicited by Ca entry through sarcolemmal voltage-gated Ca channels, a process known as Ca-induced Ca release (CICR). After activation, CICR robustly terminates due to RyR2 closure and enters a period of refractoriness during which no Ca release can be triggered. This restraining process, whose mechanistic basis has yet to be clearly defined, ensures that a substantial Ca reserve is maintained in the SR and prevents RyR2s from untimely opening during diastole. The central concept of this proposal is that genetic and acquired defects in components of the RyR2 channel are linked to a broad range of arrhythmias, encompassing catecholaminergic polymorphic ventricular tachycardia (CPVT) and post-infarction sudden cardiac death, which are associated with dysregulated SR Ca release and abnormal electrical activity. The basic pathophysiology of these arrhythmias hinges on the loss of Ca signaling stability, due to the failure of RyR2 channels to deactivate and to become appropriately refractory. A comprehensive research plan is proposed to define the fundamental processes that govern RyR2 behavior during the cardiac cycle, and how genetic and acquired defects in components of the RyR2 complex result in arrhythmogenic alterations in SR Ca release. The specific aims are to: 1) Define the mechanisms and molecular determinants of cardiac SR Ca release termination and refractoriness with a specific focus on the regulatory roles of intra-store Ca and the SR protein calsequestrin (CASQ2); 2) Define the molecular and sub-cellular determinants of CPVT linked to mutations in CASQ2 and RyR2; and 3) Define the molecular and sub-cellular mechanisms of arrhythmia associated with acquired defects in RyR2s including altered phosphorylation and redox modification. To achieve these goals, we will use state-of-the-art cellular physiology, single-channel biophysics and molecular biology approaches in combination with genetic and acquired models of cardiac disease. The findings gained from these studies will contribute to our understanding of the molecular and cellular factors involved in arrhythmias and facilitate identification of targets for rational therapies for cardiac arrhythmia. PUBLIC HEALTH RELEVANCE: Abnormalities of the heart rhythm, known as arrhythmias, are a leading cause of death in the U.S. This proposal will study how improper regulation of calcium by the muscle cells of the heart contributes to arrhythmias. Information gained from this study may help design better therapies for arrhythmias.